In silico exploration of antioxidants as oxidation protectant for PITRM1 peptidase activity, an Alzheimer disease target.

To find efficient antioxidants to protect oxidation prone cysteine residues of the peptidase PITRM1 using molecular docking and simulation techniques. A total of 50 antioxidants were docked with PITRM1 at the oxidation prone region Cys89 and Cys96 using Autodock Vina software. The lowest socred compounds were predicted for its Blood brain barrier permeability using LightBBB. Molecular dynamic simulations of the PITRM1 and Ascorbic acid/Silymarin complex were performed using the GROMACS 2020.1 package and the free energy calculations were performed using gmx_MMPBSA. The RMSD, RMSF, Rg, Minimum distance and Hydrogen bonds were also evaluated. Silymarin, Ascorbic acid, Naringenin, Gallic acid, Chlorogenic acid, Rosmarinic acid, (-)-Epicatechin, Genistein showed a docking score of above -5.3 kcal/mol. Silymarin and Ascorbic acid were predicted to cross the Blood Brain Barrier. Molecular dynamic simulation and mmPBSA analysis revealed that Silymarin showed a positive free energy implying no affinity to PITRM1 and ascorbic acid has low ΔG with -13.13 kJ/mol. The stability of the ascorbic acid complex was high (RMSD: 0.160 ± 0.018 nm, Minimum Distance: 0.163 ± 0.001 nm and four hydrogen bonds) and fluctuation induced due to ascorbic acid was low. Ascorbic acid was found to effectively interact with the cysteine oxidation prone region and can have a potential role in reducing the oxidised cysteine in PITRM1 to modulate its peptidase activity.

Expression of IDE and PITRM1 genes in ERN1 knockdown U87 glioma cells: effect of hypoxia and glucose deprivation.

OBJECTIVE: The aim of the present investigation was to study the expression of genes encoding polyfunctional proteins insulinase (insulin degrading enzyme, IDE) and pitrilysin metallopeptidase 1 (PITRM1) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of metabolism through ERN1 signaling as well as hypoxia, glucose and glutamine deprivations. METHODS: The expression level of IDE and PITRM1 genes was studied in control and ERN1 knockdown U87 glioma cells under glucose and glutamine deprivations as well as hypoxia by quantitative polymerase chain reaction. RESULTS: It was found that the expression level of IDE and PITRM1 genes was down-regulated in ERN1 knockdown (without ERN1 protein kinase and endoribonuclease activity) glioma cells in comparison with the control glioma cells, being more significant for PITRM1 gene. We also found up-regulation of microRNA MIR7-2 and MIRLET7A2, which have specific binding sites in 3'-untranslated region of IDE and PITRM1 mRNAs, correspondingly, and can participate in posttranscriptional regulation of these mRNA expressions. Only inhibition of ERN1 endoribonuclease did not change significantly the expression of IDE and PITRM1 genes in glioma cells. The expression of IDE and PITRM1 genes is preferentially regulated by ERN1 protein kinase. We also showed that hypoxia down-regulated the expression of IDE and PITRM1 genes and that knockdown of ERN1 signaling enzyme function modified the response of these gene expressions to hypoxia. Glucose deprivation increased the expression level of IDE and PITRM1 genes, but ERN1 knockdown enhanced only the effect of glucose deprivation on PITRM1 gene expression. Glutamine deprivation did not affect the expression of IDE gene in both types of glioma cells, but up-regulated PITRM1 gene and this up-regulation was stronger in ERN1 knockdown cells. CONCLUSIONS: Results of this investigation demonstrate that ERN1 knockdown significantly decreases the expression of IDE and PITRM1 genes by ERN1 protein kinase mediated mechanism. The expression of both studied genes was sensitive to hypoxia as well as glucose deprivation and dependent on ERN1 signaling in gene-specific manner. It is possible that the level of these genes expression under hypoxia and glucose deprivation is a result of complex interaction of variable endoplasmic reticulum stress related and unrelated regulatory factors and contributed to the control of the cell metabolism.

PITRM1 interaction studies with amyloidogenic nonapeptide mutants of familial Alzheimer's disease.

Amyloid ß-protein (ABP) is found to be the major cause for the development of neurodegeneration which leads to Alzheimer's. The Aß nonapeptide segment, QKLVFFAED (amino acids 15-23) is the highly amyloidogenic central region of Aß. Familial mutation in Aß increases the aggregation property of the peptide compared to the Native (Wild) amyloid-beta (Aß) and these mutations fall on the Aß nonapeptide segment. The catalytic activity of pitrilysin metallopeptidase 1(PITRM1) with familial mutant Aß (Flemish, Arctic, Dutch, Italian and Iowa) during interaction is examined using molecular dynamic simulation. The molecular dynamics simulation of PITRM1 and the Aß nonapeptide segment showed similar RMSD with respect to stability. The active site amino acid (AA) H108, hydrophobic pocket AA residues L111, F123, F124, and L127 and the basic pocket AA residues R888 and H896 showed similar interactions with both wild and familial Aß. The molecular level interaction between amyloid beta and PITRM1 were similar in the wild and familial mutants except for the Arctic mutant. The hydrophobic interaction was commonly observed between the S1 hydrophobic pocket and the LVFF region, the Arctic mutant showed less hydrogen bond formation consistently when compared to other complexes. This molecular information on catalytic activity suggests that modulating inactive PITRM1 or an increase in expression of PITRM1 can help in eliminating different kinds of familial mutant Aß in neurodegenerative cells.Communicated by Ramaswamy H. Sarma.

Exploring Whether Iron Sequestration within the CNS of Patients with Alzheimer's Disease Causes a Functional Iron Deficiency That Advances Neurodegeneration.

The involvement of iron in the pathogenesis of Alzheimer's disease (AD) may be multifaceted. Besides potentially inducing oxidative damage, the bioavailability of iron may be limited within the central nervous system, creating a functionally iron-deficient state. By comparing staining results from baseline and modified iron histochemical protocols, iron was found to be more tightly bound within cortical sections from patients with high levels of AD pathology compared to subjects with a diagnosis of something other than AD. To begin examining whether the bound iron could cause a functional iron deficiency, a protein-coding gene expression dataset of initial, middle, and advanced stages of AD from olfactory bulb tissue was analyzed for iron-related processes with an emphasis on anemia-related changes in initial AD to capture early pathogenic events. Indeed, anemia-related processes had statistically significant alterations, and the significance of these changes exceeded those for AD-related processes. Other changes in patients with initial AD included the expressions of transcripts with iron-responsive elements and for genes encoding proteins for iron transport and mitochondrial-related processes. In the latter category, there was a decreased expression for the gene encoding pitrilysin metallopeptidase 1 (PITRM1). Other studies have shown that PITRM1 has an altered activity in patients with AD and is associated with pathological changes in this disease. Analysis of a gene expression dataset from PITRM1-deficient or sufficient organoids also revealed statistically significant changes in anemia-like processes. These findings, together with supporting evidence from the literature, raise the possibility that a pathogenic mechanism of AD could be a functional deficiency of iron contributing to neurodegeneration.

Role of PITRM1 in Mitochondrial Dysfunction and Neurodegeneration.

Mounting evidence shows a link between mitochondrial dysfunction and neurodegenerative disorders, including Alzheimer Disease. Increased oxidative stress, defective mitodynamics, and impaired oxidative phosphorylation leading to decreased ATP production, can determine synaptic dysfunction, apoptosis, and neurodegeneration. Furthermore, mitochondrial proteostasis and the protease-mediated quality control system, carrying out degradation of potentially toxic peptides and misfolded or damaged proteins inside mitochondria, are emerging as potential pathogenetic mechanisms. The enzyme pitrilysin metallopeptidase 1 (PITRM1) is a key player in these processes; it is responsible for degrading mitochondrial targeting sequences that are cleaved off from the imported precursor proteins and for digesting a mitochondrial fraction of amyloid beta (Aß). In this review, we present current evidence obtained from patients with PITRM1 mutations, as well as the different cellular and animal models of PITRM1 deficiency, which points toward PITRM1 as a possible driving factor of several neurodegenerative conditions. Finally, we point out the prospect of new diagnostic and therapeutic approaches.